Method of making macroporous anion exchange resins

ABSTRACT

Methods of making macroporous anion exchange resins are described. The macroporous anion exchange resins are in the form of particles such as beads that contain a hydrophilic, crosslinked, (meth)acrylic-type polymeric material. Additionally, methods of purifying a negatively charged material using the macroporous anion exchange resins, methods of making chromatographic columns that contain the macroporous anion exchange resins, methods of making filter elements that contain the macroporous anion exchange resins, and methods of making porous composite materials that contain the macroporous anion exchange resins are described.

RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 11/326,682 filed 21,Dec. 2005.

TECHNICAL FIELD

Methods of making and using a macroporous anion exchange resin aredescribed.

BACKGROUND

Ion exchange resins are widely used within the biotechnology industryfor the large-scale separation and/or purification of various biologicalmolecules such as proteins, enzymes, vaccines, DNA, RNA, and the like.The vast majority of the anion exchange resins are based on eitherstyrene/divinylbenzene copolymers or crosslinked agarose. Thehydrophobic backbone of styrene/divinylbenzene copolymers can be proneto non-specific interactions with a number of materials leading toimpure products. Although crosslinked agarose resins are generally lesssusceptible to non-specific interactions, these materials tend to befairly soft gels and are usually unsuitable for purifications conductedwithin a chromatographic column using a high flow rate.

Some known anion exchange resins are based on (meth)acrylic-typepolymeric materials. Many of these anion exchange resins, however, aregels or have a relatively low capacity or low porosity.

SUMMARY

Methods of making macroporous anion exchange resins, methods ofpurifying a negatively charged material using the macroporous anionexchange resins, methods of making chromatographic columns that containthe macroporous anion exchange resins, methods of making filter elementsthat contain the macroporous anion exchange resins, and methods ofmaking porous composite materials that contain the macroporous anionexchange resins are described.

In one aspect, a method of forming a macroporous anion exchange resin isdescribed. The method includes preparing an aqueous phase compositionthat contains (a) a monomer mixture; (b) a water-soluble porogen ofFormula I

R¹—(R²—O)_(n)—R³  (I)

wherein R¹ is hydroxy, alkoxy, carboxy, acyloxy, or halo; each R² isindependently an alkylene having 1 to 4 carbon atoms; R³ is hydrogen,alkyl, carboxyalkyl, acyl, or haloalkyl; and n is an integer of 1 to1,000; and (c) an aqueous phase solvent mixture that includes water anda mono-alcohol having 1 to 4 carbon atoms. The mono-alcohol is presentin an amount of at least 20 weight percent based on the total weight ofthe aqueous phase solvent mixture. The monomer mixture includes (1) acrosslinking monomer containing N,N′-alkylenebis(meth)acrylamide,N,N′-heteroalkylenebis(meth)acrylamide, or a combination thereof; and(2) a positively charged ionic monomer. All of the monomers in themonomer mixture have a lipophilicity index that is less than or equal to20. The method of forming the macroporous anion exchange resin furtherincludes suspending the aqueous phase composition in a non-polar organicsolvent, polymerizing the monomer mixture to form particles of polymericmaterial, and removing the porogen from the particles.

In a second aspect, a method of separating or purifying a negativelycharged material is described. The method includes forming a macroporousanion exchange resin, contacting the macroporous anion exchange resinwith negatively charged material, and adsorbing at least a portion ofthe negatively charged material on the macroporous anion exchange resin.

In a third aspect, a method of preparing a chromatographic column isdescribed. The method includes forming a macroporous anion exchangeresin and placing the macroporous anion exchange resin in a column.

In a fourth aspect, a method of preparing a filtration element isdescribed. The method includes forming a macroporous anion exchangeresin and disposing the macroporous anion exchange resin on a surface ofa filtration medium.

In a fifth aspect, a method of making a porous composite material isdescribed. The method includes forming a macroporous anion exchangeresin and incorporating the macroporous anion exchange resin in acontinuous, porous matrix.

The above summary of the present invention is not intended to describeeach disclosed embodiment or every implementation of the presentinvention. The Figures, Detailed Description, and Examples that followmore particularly exemplify these embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention may be more completely understood in consideration of thefollowing detailed description of various embodiments of the inventionin connection with the accompanying drawings, in which:

FIG. 1 shows a scanning electron micrograph of one exemplary macroporousanion exchange resin with a magnification of 15,000×. The macroporousanion exchange resin was prepared using a polyethylene glycol porogenhaving an average molecular weight of 600 grams/mole (g/mole).

FIG. 2 shows a scanning electron micrograph of one exemplary macroporousanion exchange resin taken at with a magnification of 15,000×. Themacroporous anion exchange resin was prepared using a polyethyleneglycol porogen having an average molecular weight of 2,000 g/mole.

While the invention is amenable to various modifications and alternativeforms, specifics thereof have been shown by way of example in thedrawings and will be described in detail. It should be understood,however, that the intention is not to limit the invention to theparticular embodiments described. On the contrary, the intention is tocover all modifications, equivalents, and alternatives falling withinthe spirit and scope of the invention.

DETAILED DESCRIPTION OF THE INVENTION

Methods of making macroporous anion exchange resins are described. Themacroporous anion exchange resins are in the form of particles such asbeads that contain a hydrophilic, crosslinked, (meth)acrylic-typepolymeric material. Additionally, methods of purifying a negativelycharged material using the macroporous anion exchange resins, methods ofmaking chromatographic columns that contain the macroporous anionexchange resins, methods of making filter elements that contain themacroporous anion exchange resins, and methods of making porouscomposite materials that contain the macroporous anion exchange resinsare described.

The terms “a”, “an”, and “the” are used interchangeably with “at leastone” to mean one or more of the elements being described.

The term “alkyl” refers to a monovalent group that is a radical of analkane, which is a saturated hydrocarbon. The alkyl can be linear,branched, cyclic, or combinations thereof and typically has 1 to 20carbon atoms. In some embodiments, the alkyl group contains 1 to 10carbon atoms, 1 to 8 carbon atoms, 1 to 6 carbon atoms, or 1 to 4 carbonatoms. Examples of alkyl groups include, but are not limited to, methyl,ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl,n-hexyl, cyclohexyl, n-heptyl, n-octyl, and ethylhexyl.

The term “alkylene” refers to a divalent group that is a radical of analkane. The alkylene can be straight-chained, branched, cyclic, orcombinations thereof. The alkylene typically has 1 to 20 carbon atoms.In some embodiments, the alkylene contains 1 to 10, 1 to 8, 1 to 6, or 1to 4 carbon atoms. The radical centers of the alkylene can be on thesame carbon atom (i.e., an alkylidene) or on different carbon atoms.

The term “acyloxy” refers to a monovalent group of formula —O—(CO)—Rwhere R is an alkyl and (CO) denotes that the carbon is attached to theoxygen with a double bond. An exemplary acyloxy group is acetoxy where Ris methyl.

The term “acyl” refers to a monovalent group of formula —(CO)—R where Ris an alkyl and the (CO) denotes that the carbon is attached to theoxygen with a double bond. An exemplary acyl group is acetyl where R ismethyl.

The term “carboxy” refers to a monovalent group of formula —(CO)OH where(CO) denotes that the carbon is attached to the oxygen with a doublebond.

The term “carboxyalkyl” refers to an alkyl substituted with a carboxygroup.

The term “halo” refers to fluoro, chloro, bromo, or iodo.

The term “haloalkyl” refers to an alkyl substituted with a halo group.

The term “(meth)acrylic” refers to a polymeric material that is thereaction product of a monomer composition that includes acrylic acid,methacrylic acid, derivatives of acrylic acid or methacrylic acid, orcombinations thereof. As used herein, the term “(meth)acrylate” refersto monomers that are acrylic acid, methacrylic acid, derivatives ofacrylic acid or methacrylic acid, or combinations thereof. Suitablederivatives include esters, salts, amides, nitriles, and the like thatcan be unsubstituted or substituted. Some of these derivatives caninclude an ionic group.

The term “(meth)acryloyl” refers to a monovalent group of formulaH₂C═CR^(b)—(CO)— where R^(b) is hydrogen or methyl and (CO) denotes thatthe carbon is attached to the oxygen with a double bond.

The term “mono-alcohol” refers to an alcohol having a single hydroxygroup. The alcohol is often of formula R—OH where R is an alkyl.

The terms “polymer” or “polymeric” refer to a material that is ahomopolymer, copolymer, terpolymer, or the like. Likewise, the terms“polymerize” or “polymerization” refers to the process of making ahomopolymer, copolymer, or the like.

The term “charged” refers to a material that has an ionic group as partof its chemical structure. A negatively charged material is an anion anda positively charged material is a cation. An oppositely chargedcounterion is typically associated with the ionic group. Adjusting thepH can alter the charge of some ionic groups.

The phrase “in the range of” includes the endpoints and all numbersbetween the endpoints. For example, the phrase in the range of 1 to 10includes 1, 10, and all numbers between 1 and 10.

The term “water-soluble” used in reference to a porogen means that theporogen is miscible with the aqueous phase composition and does notpartition out of an aqueous solution into a non-polar organic solvent toany appreciable extent. For example, less than 5 weight percent, lessthan 4 weight percent, less than 3 weight percent, less than 2 weightpercent, or less than 1 weight percent of the porogen can be extractedfrom the aqueous solution into a non-polar organic solvent such as, forexample, toluene and heptane.

As used herein, the term “room temperature” refers to temperatures inthe range of 20° C. to 25° C.

Unless otherwise indicated, all numbers expressing feature sizes,amounts, and physical properties used in the specification and claimsare to be understood as being modified in all instances by the term“about.” Accordingly, unless indicated to the contrary, the numbers setforth are approximations that can vary depending upon the desiredproperties using the teachings disclosed herein.

The anion exchange resins are in the form of macroporous particles. Asused herein, the term “macroporous” refers to particles that have apermanent porous structure even in the dry state. Although the resinscan swell when contacted with a solvent, swelling is not needed to allowaccess to the interior of the particles through the porous structure. Incontrast, gel-type resins do not have a permanent porous structure inthe dry state but must be swollen by a suitable solvent to allow accessto the interior of the particles. Macroporous particles are furtherdescribed in Sherrington, Chem. Commun., 2275-2286 (1998) and Macintyreet al., Macromolecules, 37, 7628-7636 (2004).

The method of making macroporous anion exchange resins involves the useof a porogen to vary or control the average pore size, the surface area,the anion exchange capacity, or a combination thereof. The average poresize, surface area, and anion exchange capacity are variables that canalter the effectiveness of an anion exchange resin for separating and/orpurifying target molecules. Both the average pore size, which ischaracterized by the average pore diameter, and the surface area aremeasured by adsorption of nitrogen under cryogenic conditions. The anionexchange capacity refers to the amount of a negatively charged material(i.e., target molecule) that can adsorb on the anion exchange resin.Exemplary target molecules include, but are not limited to, biologicalmolecules such as proteins, enzymes, nucleic acids, and the like. Theanion exchange capacity can be given, for example, in terms of theamount of a biomolecule such as a protein (e.g., bovine serum albumin(BSA)) that can be adsorbed per unit volume of resin swollen in asolvent or per unit weight of the dry resin.

The anion exchange capacity tends to increase when the pores are largeenough to accommodate the molecules of interest (i.e., targetmolecules). The largest anion exchange capacity for biological moleculesoften can be achieved by providing a large fraction of the total surfacearea of the anion exchange resin in the form of pores that aresufficiently large to allow access of the target molecules by diffusion.

For the separation of many biological target molecules, the largestanion exchange capacity typically can be achieved when the anionexchange resin has a distribution of pore sizes. The diameters of thepores are typically in the range of about 5 nanometers to about 500nanometers. For the separation and/or purification of many biologicaltarget molecules, it is often desirous that at least some of the poresof the anion exchange resin have a diameter less than or equal to 200nanometers. The methods of preparing macroporous anion exchange resinsdescribed herein provide particles in which at least some of the poreshave a diameter less than or equal to 200 nanometers (e.g., in the rangeof 2 to 200 nanometers), less than or equal to 150 nanometers (e.g., inthe range of 2 to 150 nanometers), or less than or equal to 100nanometers (e.g., in the range of 2 to 100 nanometers).

The method of preparing the macroporous anion exchange resin includesforming an aqueous phase composition that includes (a) a monomer mixturethat contains a crosslinking monomer and a positively charged ionicmonomer; (b) a water-soluble porogen; and (c) an aqueous phase solventmixture that contains water and a mono-alcohol. The method of preparingthe macroporous anion exchange resin further includes suspending theaqueous phase composition in a non-polar organic solvent, polymerizingthe monomer mixture to form particles of polymeric material, andremoving the porogen from the anion exchange resin.

The anion exchange resins are the reaction products of a monomer mixturethat contains a crosslinking monomer and a positively charged ionicmonomer. The crosslinking monomer includesN,N′-alkylenebis(meth)acrylamide,N,N′-heteroalkylenebis(meth)acrylamide, or a combination thereof. Thepositively charged monomer has an ethylenically unsaturated groupcapable of undergoing a free radical polymerization reaction as well asa positively charged group capable of interacting with a negativelycharged material.

All or substantially all of the monomers in the monomer mixture have alipophilicity index less than or equal to 20. As used herein, the term“lipophilicity index” or “LI” refers to an index for characterizing thehydrophobic or hydrophilic character of a monomer. The lipophilicityindex is determined by partitioning a monomer in equal volumes (1:1) ofa non-polar solvent (e.g., hexane) and a polar solvent (e.g., a 75:25acetonitrile-water solution). The lipophilicity index is equal to theweight percent of the monomer remaining in the non-polar phase afterpartitioning. Monomers that are more hydrophobic tend to have a higherlipophilicity index; similarly, monomers that are more hydrophilic tendto have a lower lipophilicity index. Measurement of lipophilicity indexis further described in Drtina et al., Macromolecules, 29, 4486-4489(1996).

As used herein with reference to the lipophilicity index of the monomermixture being less than or equal to 20, the term “substantially all”means any monomer present with a lipophilicity index greater than 20 ispresent as an impurity. Any impurity with a lipophilicity index greaterthan 20 is present in an amount less than 2 weight percent, less than 1weight percent, less than 0.5 weight percent, less than 0.2 weightpercent, or less than 0.1 weight percent based on the total weight ofthe monomers in the monomer mixture. In some anion exchange resins, allor substantially all of the monomers in the monomer mixture have alipophilicity index no greater than 15, no greater than 10, no greaterthan 5, no greater than 3, or no greater than 1.

Suitable crosslinking monomers include N,N′-alkylenebis(meth)acrylamide,N,N′-heteroalkylenebis(meth)acrylamide, or a combination thereof. Thesecrosslinking monomers have at least two (meth)acryloyl groups that canreact to crosslink one polymeric chain with another polymeric chain orthat can react to crosslink one part of a polymeric chain with anotherpart of the same polymeric chain. The crosslinking monomers are solublein the aqueous phase composition and have a lipophilicity index lessthan or equal to 20.

Suitable N,N′-alkylenebis(meth)acrylamide crosslinking monomers include,but are not limited to, those having an alkylene group with 1 to 10, 1to 8, 1 to 6, or 1 to 4 carbon atoms such asN,N′-methylenebisacrylamide, N,N′-methylenebismethacrylamide,N,N′-ethylenebisacrylamide, N,N′-ethylenebismethacrylamide,N,N′-propylenebisacrylamide, N,N′-propylenebismethacrylamide,N,N′-hexamethylenebisacrylamide, andN,N′-hexamethylenebismethacrylamide. SuitableN,N′-heteroalkylenebis(meth)acrylamide crosslinking monomers include,but are not limited to, N,N′-cystaminebisacrylamide,N,N′-piperazinebisacrylamide, and N,N′-piperazinebismethacrylamide.These crosslinking monomers are commercially available from varioussuppliers such as Sigma-Aldrich (Milwaukee, Wis.) and Polysciences, Inc.(Warrington, Pa.). Alternatively, these crosslinking monomers can besynthesized by procedures described in the art such as, for example, inRasmussen, et al., Reactive Polymers, 16, 199-212 (1991/1992).

The monomer mixture includes greater than 10 weight percent crosslinkingmonomer based on the total weight of monomers in the monomer mixture.When lower amounts of the crosslinking monomer are used, the anionexchange resin tends to be in the form of a gel rather than in the formof macroporous particles. The rigidity or mechanical strength, which ismeasured by the differential pressure that the materials can withstand,tends to increase with the amount of crosslinking monomer included inthe monomer mixture. Some monomer mixtures contain greater than 10weight percent, at least 15 weight percent, at least 20 weight percent,at least 25 weight percent, or at least 30 weight percent crosslinkingmonomer.

The monomer mixture often contains up to 95 weight percent crosslinkingmonomer based on the total monomer weight. When the amount of thecrosslinking monomer exceeds 95 weight percent, the anion exchange resinoften has diminished anion exchange capacity because there is acorresponding decrease in the amount of the positively charged ionicmonomer present in the monomer mixture. Some monomer mixtures contain upto 90 weight percent, up to 85 weight percent, up to 80 weight percent,up to 70 weight percent, up to 65 weight percent, up to 60 weightpercent, up to 55 weight percent, up to 50 weight percent, up to 45weight percent, or up to 40 weight percent crosslinking monomer.

Some monomer mixtures contain greater than 10 to 95 weight percent,greater than 10 to 90 weight percent, 20 to 90 weight percent, 20 to 80weight percent, 25 to 80 weight percent, 25 to 75 weight percent, 25 to70 weight percent, 25 to 60 weight percent, or 25 to 50 weight percentcrosslinking monomer based on the total monomer weight.

The monomer mixture also includes a positively charged ionic monomer.The positively charged ionic monomer has at least one ethylenicallyunsaturated group capable of undergoing free radical polymerization. Insome embodiments, the ethylenically unsaturated group is a(meth)acryloyl group or a vinyl group. The positively charged ionicmonomer can be a weak base, a strong base, a salt of a weak base, a saltof a strong base, or combinations thereof. That is, the positivelycharged ionic monomer can be in a neutral state but capable of beingcharged if the pH is adjusted. When the pH is suitably adjusted, theresulting anion exchange resins have positively charged groups capableof interacting with negatively charged materials (i.e., anions). If theionic monomer used to prepare a anion exchange resin includes a salt ofa weak base or a salt of a strong base, the counter ions of these saltscan be, but are not limited to, a halide (e.g., chloride), a carboxylate(e.g., acetate or formate), nitrate, phosphate, sulfate, bisulfate,methyl sulfate, or hydroxide.

Some exemplary ionic monomers are of amino(meth)acrylates or amino(meth)acrylamides of Formula II or quaternary ammonium salts thereof.

In Formula II, R⁴ is hydrogen or methyl; L is oxy or —NH—; and Y is analkylene (e.g., an alkylene having 1 to 10 carbon atoms, 1 to 6, or 1 to4 carbon atoms). Each R⁵ is independently hydrogen, alkyl, hydroxyalkyl(i.e., an alkyl substituted with a hydroxy), or aminoalkyl (i.e., analkyl substituted with an amino). Alternatively, the two R⁵ groups takentogether with the nitrogen atom to which they are attached can form aheterocyclic group that is aromatic, partially unsaturated (i.e.,unsaturated but not aromatic), or saturated, wherein the heterocyclicgroup can optionally be fused to a second ring that is aromatic (e.g.,benzene), partially unsaturated (e.g., cyclohexene), or saturated (e.g.,cyclohexane). The counter ions of the quaternary ammonium salts areoften halides, sulfates, phosphates, nitrates, and the like.

In some embodiments of Formula II, both R⁵ groups are hydrogen. In otherembodiments, one R⁵ group is hydrogen and the other is an alkyl having 1to 10, 1 to 6, or 1 to 4 carbon atoms. In still other embodiments, atleast one of R⁵ groups is a hydroxy alkyl or an amino alkyl that have 1to 10, 1 to 6, or 1 to 4 carbon atoms with the hydroxy or amino groupbeing positioned on any of the carbon atoms of the alkyl group. In yetother embodiments, the R⁵ groups combine with the nitrogen atom to whichthey are attached to form a heterocyclic group. The heterocyclic groupincludes at least one nitrogen atom and can contain other heteroatomssuch as oxygen or sulfur. Exemplary heterocyclic groups include, but arenot limited to imidazolyl. The heterocyclic group can be fused to anadditional ring such as a benzene, cyclohexene, or cyclohexane.Exemplary heterocyclic groups fused to an additional ring include, butare not limited to, benzoimidazolyl.

Exemplary amino(meth)acrylates (i.e., L in Formula II is oxy) includeN,N-dialkylaminoalkyl(meth)acrylates such as, for example,N,N-dimethylaminoethylmethacrylate, N,N-dimethylaminoethylacrylate,N,N-diethylaminoethylmethacylate, N,N-diethylaminoethylacrylate,N,N-dimethylaminopropylmethacrylate, N,N-dimethylaminopropylacrylate,N-tert-butylaminopropylmethacrylate, N-tert-butylaminopropylacrylate andthe like.

Exemplary amino(meth)acrylamides (i.e., L in Formula II is —NH—)include, for example, N-(3-aminopropyl)methacrylamide,N-(3-aminopropyl)acrylamide, N-[3-(dimethylamino)propyl]methacrylamide,N-(3-imidazolylpropyl)methacrylamide, N-(3-imidazolylpropyl)acrylamide,N-(2-imidazolylethyl)methacrylamide,N-(1,1-dimethyl-3-imidazoylpropyl)methacrylamide,N-(1,1-dimethyl-3-imidazoylpropyl)acrylamide,N-(3-benzoimidazolylpropyl)acrylamide, andN-(3-benzoimidazolylpropyl)methacrylamide.

Exemplary quaternary salts of the ionic monomers of Formula II include,but are not limited to, (meth)acrylamidoalkyltrimethylammonium salts(e.g., 3-methacrylamidopropyltrimethylammonium chloride and3-acrylamidopropyltrimethylammonium chloride) and(meth)acryloxyalkyltrimethylammonium salts (e.g.,2-acryloxyethyltrimethylammonium chloride,2-methacryloxyethyltrimethylammonium chloride,3-methacryloxy-2-hydroxypropyltrimethylammonium chloride,3-acryloxy-2-hydroxypropyltrimethylammonium chloride, and2-acryloxyethyltrimethylammonium methyl sulfate).

Other monomers that can provide positively charged groups to an ionexchange resin include the dialkylaminoalkylamine adducts ofalkenylazlactones (e.g., 2-(diethylamino)ethylamine,(2-aminoethyl)trimethylammonium chloride, and3-(dimethylamino)propylamine adducts of vinyldimethylazlactone) anddiallylamine monomers (e.g., diallylammonium chloride anddiallyldimethylammonium chloride).

The monomer mixture includes at least 5 weight percent of the positivelycharged monomer based on the total weight of monomers in the monomermixture. When lower levels of the positively charged monomer are used,the anion exchange resin often has diminished anion exchange capacity.Some monomer mixtures contain at least 10 weight percent, at least 15weight percent, at least 20 weight percent, at least 25 weight percent,at least 30 weight percent, or at least 35 weight percent of thepositively charged monomer.

The monomer mixture often contains less than 90 weight percent of thepositively charged ionic monomer based on the total weight of themonomers. When higher levels of the positively charged ionic monomer areused, the anion exchange resins tend to be gels rather than macroporousparticles. That is, higher levels of positively charged ionic monomersare often accompanied by a corresponding decrease in the amount ofcrosslinking monomer. The rigidity and mechanical strength of the anionexchange resin tends to correlate with the amount of crosslinkingmonomer. Some monomer mixtures contain less than 90 weight percent, nogreater than 85 weight percent, no greater than 80 weight percent, nogreater than 75 weight percent, no greater than 70 weight percent, nogreater than 65 weight percent, no greater than 60 weight percent, nogreater than 55 weight percent, no greater than 50 weight percent, nogreater than 45 weight percent, or no greater than 40 weight percent ofthe positively charged monomer.

Some monomer mixtures contain 5 to less than 90 weight percent, 10 toless than 90 weight percent, 20 to less than 90 weight percent, 30 toless than 90 weight percent, 30 to 80 weight percent, 40 to less than 90weight percent, 40 to 80 weight percent, 50 to 80 weight percent, or 60to 80 weight percent of the positively charged monomer based on thetotal monomer weight. The amount of positively charged monomer andcrosslinking monomer can be balanced to provide an anion exchange resinwith the desired combination of anion exchange capacity and mechanicalstrength.

For applications such as those that include the use of the macroporousion exchange resins within a column, monomer mixtures containing 25 to75 weight percent of the ionic monomer and 25 to 75 weight percent ofthe crosslinking monomer often provide the best balance of anionexchange capacity and mechanical strength. Some exemplary monomermixtures include 35 to 75 weight percent of the positively charged ionicmonomer and 25 to 65 weight percent of the crosslinking monomer, 40 to75 weight percent of the positively charged ionic monomer and 25 to 60weight percent of the crosslinking monomer, 50 to 75 weight percent ofthe positively charged ionic monomer and 25 to 50 weight percent of thecrosslinking monomer, or 60 to 70 percent of the positively chargedionic monomer and 30 to 40 weight percent of the crosslinking monomer.

In some specific monomer mixtures, the crosslinking monomer is aN,N′-alkylenebis(meth)acrylamide and the positively charged ionicmonomer is of Formula II. The monomer mixture includes 25 to 75 weightpercent of the positively charged ionic monomer and 25 to 75 weightpercent of the crosslinking monomer, 35 to 75 weight percent of thepositively charged ionic monomer and 25 to 65 weight percent of thecrosslinking monomer, 40 to 75 weight percent of the positively chargedionic monomer and 25 to 60 weight percent of the crosslinking monomer,50 to 75 weight percent of the positively charged ionic monomer and 25to 50 weight percent of the crosslinking monomer, or 60 to 70 percent ofthe positively charged ionic monomer and 30 to 40 weight percent of thecrosslinking monomer. For example, the monomer mixture can contain 35weight percent of the crosslinking monomer and 65 weight percent of thepositively charged ionic monomer.

Although some monomer mixtures are free of monomers other than thecrosslinking monomer and the positively charged ionic monomer, othermonomer mixtures include a hydrophilic but non-ionic monomer having asingle ethylenically unsaturated group. The hydrophilic, non-ionicmonomer can be added, for example, for the purpose of adjusting theanion exchange capacity while maintaining the amount of crosslinkingmonomer constant. That is, the anion exchange capacity can be modifiedwithout significantly altering the amount of crosslinking, or therigidity of the anion exchange resin. Additionally, the hydrophiliccharacter of the anion exchange resins can be modified with the use ofthese non-ionic monomers.

Suitable hydrophilic, non-ionic monomers are typically present inamounts no greater than 50 weight percent based on the total weight ofthe monomers in the monomer mixture. In some anion exchange resins, themonomer mixture contains no greater than 40 weight percent, no greaterthan 20 weight percent, no greater than 10 weight percent, no greaterthan 5 weight percent, no greater than 2 weight percent, or no greaterthan 1 weight percent hydrophilic, non-ionic monomer based on the totalweight of monomers.

Examples of non-ionic monomers that have a sufficiently lowlipophilicity index include, but are not limited to,hydroxyalkyl(meth)acrylates such as 2-hydroxyethylacrylate,3-hydroxypropylacrylate, 2-hydroxyethylmethacrylate (e.g., LI is 1), and3-hydroxypropylmethacrylate (e.g., LI is 2); acrylamide (e.g., LI isless than 1) and methacrylamide(LI is less than 1); glycerolmonomethacrylate and glycerol monoacrylate; N-alkyl(meth)acrylamidessuch as N-methylacrylamide (e.g., LI is less than 1),N,N-dimethylacrylamide (e.g., LI is less than 1),N-methylmethacrylamide, and N,N-dimethylmethacrylamide; N-vinylamidessuch as N-vinylformamide, N-vinylacetamide, and N-vinylpyrrolidone;acetoxyalky(meth)acrylates such as 2-acetoxyethylacrylate and2-acetoxyethylmethacrylate (e.g., LI is 9); glycidyl(meth)acrylates suchas glycidylacrylate and glycidylmethacrylate (e.g., LI is 11); andvinylalkylazlactones such as vinyldimethylazlactone (e.g., LI is 15).

The monomer mixture is generally substantially free of hydrophobicmonomers. More specifically, the monomer mixture is substantially freeof monomers having a lipophilicity index greater than 20. Anionicexchange resins that are substantially free of hydrophobic monomers tendto have low non-specific adsorption of impurities such as non-targetmaterials such as non-target proteins, lipids, and the like. Monomersthat have a lipophilicity index greater than 20 and that are generallynot in the monomer mixture include, for example,ethyleneglycoldimethacrylate (LI is 25), phenoxyethylmethacrylate (LI is32), trimethylolpropanetrimethacrylate (LI is 37), methylmethacrylate(LI is 39), ethylmethacrylate (LI is 53), butylmethacrylate (LI is 73),cyclohexylmethacrylate (LI is 90), laurylmethacrylate (LI is 97), andthe like.

The aqueous phase composition usually contains at least 4 weight percentmonomer mixture based on the total weight of the aqueous phasecomposition e.g., monomer mixture, porogen, and aqueous phase solventmixture). In some embodiments, the aqueous phase composition can containat least 10 weight percent or at least 15 weight percent monomermixture. The aqueous phase composition usually contains up to 50 weightpercent monomer mixture based on the total weight of the aqueous phasecomposition. In some embodiments, the aqueous phase composition cancontain up to 40 weight percent, up to 30 weight percent, up to 25weight percent, or up to 20 weight percent monomer mixture. For example,the aqueous phase composition can contain 5 to 30 weight percent, 5 to25 weight percent, 5 to 20 weight percent, or 10 to 20 weight percentmonomer mixture based on the total weight of the aqueous phasecomposition.

In addition to the monomer mixture, the aqueous phase compositionincludes a water-soluble porogen. The porogen facilitates the formationof a macroporous anion exchange resin that can be used for thepurification and/or separation of biological molecules. The porogen isnot a monomer and is free of groups such as ethylenically unsaturatedgroups that can undergo a free radical polymerization reaction witheither the crosslinking monomer or the positively charged ionic monomer.The porogen, in general, is not covalently attached to the polymericmaterial and is usually removed after the polymerization reaction iscomplete. During the polymerization reaction, however, the porogen maycovalently bond to the polymeric material of the anion exchange resinthrough a chain transfer reaction. Preferably, the porogen is not bondedto the polymeric material of the anion exchange resin. The porogen is analkylene oxide or polyalkylene oxide of Formula I

R¹—(R²—O)_(n)—R³  (I)

wherein R¹ is hydroxy, alkoxy, carboxy, acyloxy, or halo; each R² isindependently an alkylene having 1 to 4 carbon atoms; R³ is hydrogen,alkyl, carboxyalkyl, acyl, or haloalkyl; and n is an integer of 1 to1,000.

In some exemplary porogens, both end groups (i.e., group —R¹ and group—OR³) are hydroxy groups (i.e., R¹ is hydroxy and R³ is hydrogen). Inother exemplary porogens, R¹ is hydroxy and R³ is an alkyl (e.g., analkyl having 1 to 20, 1 to 10, 1 to 6, or 1 to 4 carbon atoms),haloalkyl (e.g., chloroalkyl such as chloromethyl), acyl (e.g., acetyl),or carboxyalkyl (e.g., carboxymethyl). That is, one end group is hydroxyand the other end group is an alkoxy, haloalkoxy, acyloxy, or carboxy(e.g., carboxyalkoxy, which is an alkoxy substituted with a carboxy). Inother exemplary porogens, R¹ is an alkoxy (e.g., an alkoxy having 1 to10, 1 to 6, or 1 to 4 carbon atoms) and R³ is an alkyl (e.g., an alkylhaving 1 to 10, 1 to 6, or 1 to 4 carbon atoms) or an acyl (e.g.,acetyl). That is, one end group is an alkoxy and the other end group isan alkoxy or acyloxy. In still other exemplary porogens, R¹ is carboxyand R³ is carboxy alkyl (e.g., carboxymethyl). That is, both end groupsare carboxy (—OR³ is carboxyalkoxy).

Group R² in Formula I is an alkylene such as, for example, methylene,ethylene, or propylene. Suitable porogens with ethylene R² groupsinclude ethylene glycol and ethylene glycol based materials such asdiethylene glycol, triethylene glycol, and higher homologs. The higherhomologs of ethylene glycol are often referred to as polyethylene glycol(i.e., PEG) or polyethylene oxide (i.e., PEO). Other suitable porogenswith propylene R² groups include propylene glycol and propylene glycolbased materials such as dipropylene glycol, tripropylene glycol, andhigher homologs. The higher homologs of propylene glycol are oftenreferred to as polypropylene glycol (i.e., PPG) or polypropylene oxide(i.e., PPO). The porogens can be random or block copolymers ofpolyethylene oxide and polypropylene oxide.

Subscript n in Formula I can be an integer greater than 1, greater than2, greater than 5, greater than 10, greater than 20, greater than 50,greater than 100, greater than 200, or greater than 500. For example, ncan be an integer in the range of 1 to 1,000, in the range of 1 to 800,in the range of 1 to 600, in the range of 1 to 500, in the range of 1 to200, or in the range of 1 to 100.

Some porogens are polyalkylene oxides having a molecular weight of atleast 200 g/mole, at least 400 g/mole, at least 800 g/mole, at least1,000 g/mole, at least 2,000 g/mole, 4,000 g/mole, at least 8,000g/mole, or at least 10,000 g/mole. The polyalkylene oxide porogens oftenhave an average molecular weight up to 20,000 g/mole, up to 16,000g/mole, up to 12,000 g/mole, up to 10,000 g/mole, up to 8,000 g/mole, upto 6,000 g/mole up to 4,000 g/mole, up to 2,000 g/mole, or up to 1,000g/mole. For example, the polyalkylene oxide porogen typically has anaverage molecular weight in the range of 200 to 20,000 g/mole, in therange of 200 to 16,000 g/mole, in the range of 200 to 8,000 g/mole, inthe range of 200 to 4,000 g/mole, in the range of 200 to 2,000 g/mole,or in the range of 200 to 1,000 g/mole.

In some methods of forming the anion exchange resin, a mixture ofporogens can be used. For example, the porogen can be a mixture of afirst porogen being alkylene glycol (i.e., n is equal to 1 in Formula I)and a second porogen that is a polyalkylene oxide (i.e., n is greaterthan 1 in Formula I). In a more specific example, the porogen can be amixture of ethylene glycol with a polyethylene oxide with hydroxy endgroups.

Polyalkylene oxides are commercially available that have end groups(i.e., groups —R¹ and —OR³) selected from hydroxy, methoxy, acombination of hydroxy and methoxy, a combination of hydroxy and chloro,a combination of alkoxy and acetoxy, or at least one carboxy group. Suchmaterials can be obtained, for example, from Sigma-Aldrich (Milwaukee,Wis.), Nektar (Huntsville, Ala.), and Dow Chemical (Midland, Mich.).

The method of preparing the anion exchange resin is an inversesuspension polymerization process. The aqueous phase composition, whichincludes (a) the monomer mixture, (b) the porogen, and (c) the aqueousphase solvent mixture, is dispersed or suspended as droplets in anon-polar organic solvent with the volume of the non-polar organicsolvent typically being greater than the volume of the aqueous phasecomposition. The porogen is usually a liquid that can function as aconventional solvent for the monomer mixture within the aqueous phasecomposition. Useful porogens generally do not partition between theaqueous phase composition and the non-polar organic solvent to anyappreciable extent (i.e., the porogen is not extracted in anyappreciable amount from the aqueous phase composition into the non-polarsolvent). For example, less than 5 weight percent, less than 4 weightpercent, less than 3 weight percent, less than 2 weight percent, or lessthan 1 weight percent of the porogen can be extracted from the aqueousphase composition into the non-polar organic solvent such as toluene,heptane, or the like.

As the free radical polymerization reaction proceeds, many polymericmolecules are formed within each aqueous phase droplet. The polymericmolecules continue to grow and crosslink as the reaction proceeds. Whenthe molecular weight becomes sufficiently large, a polymeric phaseseparates from the aqueous phase composition within the droplet.Although not wanting to be bound by theory, it is believed that thepores are formed, at least in part, by the exclusion of the aqueoussolvent from the polymeric material as the molecular weight increases.The point at which phase separation occurs can influence the averagepore size and the pore size distribution. A later phase separation tendsto favor the formation of anion exchange resin particles that havesmaller pores and larger surface areas. Conversely, an earlier phaseseparation tends to favor the formation of anion exchange resinparticles that have larger pores and smaller surface areas.

The point at which phase separation occurs can be influenced by thecompatibility of the porogen with the forming polymeric material and theamount of porogen. Additionally, the point at which phase separationoccurs can be influenced by the amount of crosslinking monomer presentin the monomer mixture, with larger amounts of crosslinking monomertypically favoring earlier phase separation due to a more rapid increasein the molecular weight of the polymeric material.

Porogens that are compatible with the forming polymeric material (i.e.,porogens that are good solvents for the forming polymeric material) tendto result in a later phase separation compared to porogens that are lesscompatible with the forming polymeric material (porogens that are poorsolvents for the forming polymeric material). Porogens with a highersolubility for the forming polymeric material tend to result in theformation of anion exchange resin particles that have smaller pores andlarger surface areas compared to porogens having a lower solubility forthe forming polymeric material. Conversely, porogens with a lowersolubility for the forming polymeric material tend to result in theformation of anion exchange particles that have larger pores and smallersurface areas compared to porogens having a greater solubility for theforming polymeric material.

The anion exchange resin can be designed for the target molecule ofinterest. That is, the anion exchange resin can be designed to optimizethe anion exchange capacity for a particular target molecule. Anionexchange resins with larger pore sizes are often more suitable forlarger target molecules. The molecular weight of the porogen, the endgroup of the porogen (i.e., groups —R¹ and —OR³ in Formula I), theamount of the porogen, and the composition of the monomer mixture canaffect the average pore size, surface area, and pore volume.

The addition of porogen tends to increase the surface area, pore size,and the anion exchange capacity (i.e., capacity for a target molecule)until a maximum binding capacity is reached for the particular choice ofporogen and monomer composition. Further addition of porogen can eitherresult in no change in the anion exchange capacity or can result in adecreased anion exchange capacity. A decreased anion exchange capacityis typically accompanied by a decrease in the surface area and anincrease in the average pore size. As used herein, the term “maximumbinding capacity” refers to the maximum anion exchange capacity for aparticular target molecule and for a particular combination of porogenand monomer composition.

With porogens having hydroxy end groups and an average molecular weightup to about 1,000 g/mole, macroporous anion exchange resins can beprepared using, for example, up to 100 weight percent or more porogenbased on the weight of monomers in the monomer mixture. Exemplary anionexchange resins can be prepared using up to 80 weight percent, up to 60weight percent, up to 40 weight percent, up to 20 weight percent, or upto 10 weight percent porogen based on the weight of monomers in themonomer mixture.

If a polyethylene oxide with hydroxy end groups and an average molecularweight up to about 1,000 g/mole is used as a porogen, the porogenconcentration is typically in the range of 0.1 to 20 weight percentbased on the weight of the aqueous phase composition (e.g., the combinedweight of the monomer mixture, porogen, and aqueous solvent mixture).For example, the amount of porogen can be in the range of 0.2 to 20weight percent, 0.3 to 20 weight percent, 0.3 to 15 weight percent, 0.5to 15 weight percent, 1 to 15 weight percent, or 2 to 15 weight percentbased on the total weight of the aqueous phase composition.

Porogens with an average molecular weight greater than about 1,000g/mole are typically used in a lower amount than porogens with a loweraverage molecular weight. With these higher molecular weight porogens,increasing the amount of porogen in the aqueous phase composition beyondthe amount needed for maximum binding capacity often tends to increasethe average pore size, decrease the surface area, and decrease the anioncapacity. That is, the average pore size, the surface area, and theanion exchange capacity often cannot be maintained with the addition oflarger amounts of porogen.

Porogens with alkoxy end groups are typically used in lower amounts thanporogens with hydroxy end groups. Porogens with alkoxy end groups tendto be less soluble in an aqueous phase composition and less compatiblewith the forming polymeric material compared to porogens with hydroxyend groups. With these porogens having alkoxy end groups, increasing theamount of porogen in the aqueous phase composition beyond the amountneeded for maximum binding capacity often tends to increase the averagepore size, decrease the surface area, and decrease the anion exchangecapacity. That is, the average pore size, the surface area, and theanion exchange capacity cannot be maintained with the addition of largeramounts of porogen.

The aqueous phase composition contains an aqueous solvent mixture thatincludes water plus a mono-alcohol having 1 to 4 carbon atoms. Suitablemono-alcohols include methanol, ethanol, n-propanol, iso-propanol,tert-butanol, or a combination thereof. The mono-alcohol contributes, atleast partially, to the total porosity of the macroporous anion exchangeresins. That is, the porosity of the ion exchange resins tends to behigher with the addition of the mono-alcohol (i.e., the mono-alcohol canfunction as both a solvent and as a co-porogen).

At least 20 weight percent of the aqueous phase solvent mixture can be amono-alcohol. In some embodiments, at least 30 weight percent, at least40 weight percent, or at least 50 weight percent of the aqueous phasesolvent mixture can be the mono-alcohol. Up to 80 weight percent, up to70 weight percent, or up to 60 weight percent of the aqueous phasesolvent mixture can be the mono-alcohol. For example, the amount ofmono-alcohol can be in the range of 20 to 80 weight percent, 30 to 80weight percent, 40 to 80 weight percent, or 50 to 80 weight percent ofthe aqueous phase solvent mixture.

The aqueous phase solvent mixture can also contain additionalco-solvents that are miscible with water and the mono-alcohol. Suitableaqueous phase co-solvents include, but are not limited to,dimethylsulfoxide, dimethylformamide, N-methylpyrrolidone, acetonitrile,and the like. The co-solvent can, for example, improve the solubility ofsome of the monomers such as the crosslinking monomer in the aqueousphase composition. The co-solvent can influence the phase separationbehavior of the forming polymer, and will influence the porositycharacteristics of the resultant anion exchange resin.

The aqueous phase composition is dispersed or suspended in a non-polarorganic solvent. The volume ratio of non-polar organic solvent to theaqueous phase composition is usually in the range of 2:1 to 6:1. Theaqueous phase composition, which contains the monomer mixture andporogen, is often dispersed as relatively small droplets in thenon-polar organic solvent. Besides functioning as an inert medium fordispersion of the aqueous phase composition, the primary purpose of thesuspending medium (i.e., the non-polar organic solvent) is to dissipatethe heat generated during the polymerization reaction. In someembodiments, the density of the suspension medium can be selected to beapproximately the same as the aqueous phase composition. Approximatelymatching these densities tends to result in the formation of morespherical particles as well as more uniformly sized particles.

Suitable non-polar organic solvents are typically alkanes such ashexane, heptane, n-octane, isooctane, isododecane, and cyclohexane;halogenated hydrocarbons such as carbon tetrachloride, chloroform, andmethylene chloride; aromatics such as benzene and toluene; low-viscositysilicone oils; or combinations thereof. For example, the non-polarorganic solvent can be a mixture of heptane and methylene chloride orheptane and toluene.

A suspending agent (i.e., polymeric stabilizer) is often used tofacilitate suspension of the aqueous phase composition droplets in thenon-polar organic solvent. Unlike the porogen that is hydrophilic, thesuspending agent usually has both hydrophobic and hydrophilic portions.The suspending agent functions to modify the interfacial tension betweenthe aqueous phase composition and the non-polar organic solvent.Additionally, the suspending agent provides steric stabilization of theaqueous phase composition droplets. This steric stabilization tends tominimize or prevent the formation of agglomerated particles during thepolymerization process.

Suitable suspending agents include sorbitan sesquioleate, polyethyleneoxide (20) sorbitan trioleate, polyethylene oxide (20) sorbitanmonooleate, sorbitan trioleate, sodium di-2-ethylhexylsulfosuccinate, acopolymer of isooctylacrylate and acrylic acid, a copolymer ofhexylacrylate and sodium acrylate, a copolymer of isooctylacrylate and2-acrylamidoisobutyramide, and the like. The amount of suspending agentcan influence the size of the anion exchange resin (i.e., the use oflarger amounts of suspending agent often results in the formation ofsmaller anion exchange resin particles). The amount of the suspendingagent is generally 0.1 to 10 weight percent based on the total weight ofthe monomers in the monomer mixture. For example, the monomer mixturecan contain 0.1 to 8 weight percent or 0.5 to 5 weight percentsuspending agent based on the total weight of monomers.

The size of the anion exchange resin is determined, to a large extent,by the size of the aqueous phase composition droplets. The droplet sizecan be affected by variables such as the rate of agitation, thetemperature, the amount of suspending agent, the choice of suspendingagent, the choice of non-polar organic solvent, and the choice of anyaqueous phase co-solvents. The rate of agitation, the type of suspendingagent, and the amount of suspending agent can often be varied to controlthe aggregation or agglomeration of the resulting particles. A lack ofaggregation is generally preferred.

An initiator can be added to the aqueous phase composition to commencethe free radical polymerization reaction. The free radical initiator isusually soluble in the aqueous phase solvent mixture. Once thesuspension has been formed, the free radical initiator can be activatedthermally, photochemically, or through an oxidation-reduction reaction.The free radical initiator is often used in an amount of 0.02 to 10weight percent based on the total weight of the monomers in the monomermixture. In some examples, the free radical initiator is present in anamount of 2 to 6 weight percent based on the total weight of themonomers.

Suitable water-soluble thermal initiators include, for example, azocompounds, peroxides or hydroperoxides, persulfates, and the like.Exemplary azo compounds include2,2′-azobis[2-(2-imidazolin-2-yl)propane]dihydrochloride,2,2′-azobis(2-amidinopropane)dihydrochloride, and4,4′-azobis-(4-cyanopentanoic acid). Examples of commercially availablethermal azo compound initiators include materials available from DuPontSpecialty Chemical (Wilmington, Del.) under the “VAZO” trade designationsuch as VAZO 44, VAZO 56, and VAZO 68. Suitable peroxides andhydroperoxides include acetyl peroxide, tert-butyl hydroperoxide, cumenehydroperoxide, and peroxyacetic acid. Suitable persulfates include, forexample, sodium persulfate and ammonium persulfate.

In other examples, the free radical initiator is a redox couple such asammonium or sodium persulfate and N,N,N′,N′-tetramethylethylenediamine(TMEDA); ammonium or sodium persulfate and ferrous ammonium sulfate;hydrogen peroxide and ferrous ammonium sulfate; cumene hydroperoxide andN,N-dimethylaniline; or the like. The polymerization temperaturetypically depends on the specific free radical initiator chosen and onthe boiling point of the non-polar organic solvent. The polymerizationtemperature is usually about 50° C. to about 150° C. for thermallyinitiated polymerizations. In some methods, the temperature is about 55°C. to about 100° C. For redox or photochemically initiatedpolymerizations, the temperature can be close to room temperature orbelow, if desired. The polymerization time can be about 30 minutes toabout 24 hours or more. Typically, a polymerization time of 2 to 4 hoursis sufficient.

Once the free radical polymerization reaction has been initiated, theforming polymeric material tends to precipitate within the aqueous phasecomposition. Some of the aqueous phase composition such as the porogencan get trapped in the pores of polymeric material. The particles ofpolymeric material can be isolated, for example, by filtration ordecantation. The particles of polymeric material can then be subjectedto a series of washing steps to remove the porogen. Suitable solventsfor removing the porogen include polar solvents such as, for example,water, acetone, alcohols (e.g., methanol, ethanol, n-propanol, andiso-propanol), dimethylsulfoxide, dimethylformamide,N-methylpyrrolidone, acetonitrile, and the like. The resulting anionexchange resins can be dried using any suitable method, if desired. Insome methods, the resulting anion exchange resins can be fractionatedusing techniques such as screening, sedimentation, and airclassification.

The macroporous anion exchange resin particles can have an irregularshape or can be spherical or roughly spherical. In some examples, themacroporous anion exchange resin particles are beads. The average sizeof the particles can be determined using techniques such as lightscattering or electron microscopy with image analysis. The particlesusually have an average size of at least 10 micrometers. For example,the particles can have an average size of at least 20 micrometers, atleast 30 micrometers, at least 40 micrometers, at least 50 micrometers,or at least 60 micrometers. The particles usually have an average sizeno greater than 2,000 micrometers, no greater than 1,000 micrometers, nogreater than 500 micrometers, or no greater than 200 micrometers. Insome applications, the macroporous anion exchange resins have an averageparticle size of 10 to 2,000 micrometers, 20 to 2,000 micrometers, 20 to500 micrometers, 50 to 500 micrometers, 20 to 200 micrometers, 50 to 200micrometer, 50 to 100 micrometers, 50 to 75 micrometers, 50 to 70micrometers, or 60 to 70 micrometers.

If the average size of the macroporous anion exchange resin particles isless than about 10 micrometers or less than about 20 micrometers, thenthe back pressure in a chromatographic column filled with the particlesmay become unacceptably large, especially for the large columns (e.g.,columns with a diameter greater than about 5 cm) that can be used forthe purification or separation of large biological molecules. Althoughthe average particle size can be as large as 2,000 micrometers, theaverage particle size for some applications (e.g., applications in whichthe macroporous anion exchange resins are placed in large columns) isoften no greater than 200 micrometers. If the average particle size islarger, the efficiency of the chromatographic process may be low,especially for the purification or separation of large biologicalmolecules such as proteins that often have low diffusion rates into thepores of the macroporous anion exchange resin. For example, to achievethe same degree of separation or purity with larger anion exchangeresins that can be obtained using anion exchange resins of 20 to 200micrometers, a greater amount of the resin, a longer chromatographiccolumn, a slower flow rate, or a combination thereof may be needed.

The porosity and surface area of the anion exchange resin particles canbe characterized by adsorbing nitrogen onto the surface of the particlesat various relative pressures under cryogenic conditions (i.e., a sampleof the anion exchange resin within a tube is subjected to a vacuum andthe tube is placed in liquid nitrogen for cooling). Nitrogen is adsorbedon the surface of the sample at multiple relative pressures (e.g., fromabout 0.0 to about 1.0) and then desorbed at multiple relativepressures. BJH theory, which is further described in E. P. Barrett, L.S. Joyner, and P. P. Halenda, J. Am. Chem. Soc., 73, 373 (1951), can beused to relate the amount of nitrogen adsorbed or desorbed at themultiple relative pressures to pores having pore diameters in the rangeof about 2 to about 200 nanometers. The pore volume, surface area, andaverage pore diameter can be calculated. As used herein, the term “porevolume” refers to the cumulative pore volume calculated using BJH theoryfrom the adsorption of nitrogen at various relative pressures from about0.0 to about 1.0. As used herein, the term “surface area” refers to thecumulative surface area calculated using BJH theory from the adsorptionof nitrogen at various relative pressures from about 0.0 to about 1.0.As used herein, the term “average pore diameter” is the average porediameter measured using BJH theory from the adsorption of nitrogen atvarious relative pressures from about 0.0 to about 1.0.

The macroporous anion exchange resins have a distribution of pore sizes.The pore diameters can be up to 500 nanometers or larger. The anionexchange resins have pores in the size range that can be measured usingnitrogen adsorption techniques. That is, at least some of the pores havea diameter less than 200 nanometers, less than 150 nanometers, or lessthan 100 nanometers. The average pore diameter measured by nitrogenadsorption is typically at least 2 nanometers, at least 5 nanometers, atleast 10 nanometers, at least 20 nanometers, or at least 30 nanometers.The average pore diameter can be up to 200 nanometers, up to 100nanometers, or up to 80 nanometers. For example, the average porediameters can be in the range of 10 to 200 nanometers, in the range of10 to 100 nanometers, in the range of 10 to 80 manometers, in the rangeof 20 to 100 nanometers, or in the range of 20 to 80 nanometers.

The pore volume is often at least 0.10 cubic centimeters per gram. Forexample, the pore volume can be at least 0.15 cubic centimeters pergram, at least 0.20 cubic centimeters per gram, or at least 0.25 cubiccentimeters per gram. The pore volume can be in the range of 0.10 to 2cubic centimeters per gram, in the range of 0.15 to 2 cubic centimetersper gram, or in the range of 0.2 to 2 cubic centimeters per gramresulting from pores having a diameter no greater than 200 nanometers.The pores are large enough to accommodate the biological materials butsmall enough to provide adequate surface area and anion exchangecapacity.

The surface area is usually at least 20 m²/g, at least 30 m²/g, or atleast 40 m²/g. The surface area is often in the range of 20 to 200 m²/g,in the range of 30 to 200 m²/g, in the range of 20 to 100 m²/g, in therange of 30 to 100 m²/g, or in the range of 40 to 200 m²/g.

The anion exchange capacity of an anion exchange resin can be given interms of the amount of a protein such as bovine serum albumin (BSA) thatcan be adsorbed. More particularly, some anion exchange resins have abovine serum albumin anion exchange capacity that is at least 5 mg/mL(i.e., 5 milligrams of bovine serum albumin per milliliter of anionexchange resin). For example, some anion exchange resins have a bovineserum albumin anion exchange capacity that is at least 15 mg/mL, atleast 20 mg/mL, at least 25 mg/mL, at least 30 mg/mL, or at least 40mg/mL. More particularly, some anion exchange resins have a bovine serumalbumin anion exchange capacity of 5 to 100 mg/mL, 5 to 70 mg/mL, 20 to70 mg/mL, 20 to 60 mg/mL, 30 to 60 mg/mL, or 30 to 50 mg/mL.

The anion exchange resins prepared using the methods described hereinare hydrophilic and usually have a low non-specific adsorption (i.e.,anion exchange resins prepared from monomer with low LI tend to have lownon-specific adsorption). The anion exchange resins typically adsorbvarious negatively charged materials through interaction with thepositively charged groups on the anion exchange resin and typicallyadsorb little, if any, material on the non-ionic portions of the anionexchange resin. This low non-specific adsorption can advantageouslyresult in better separation or purification of negatively charged targetmaterials from other materials in a sample. In some examples, thecharged target materials are impurities such as anionic proteins,nucleic acids, endotoxins, lipids, phospholipids, or the like that needto be removed from a protein solution (e.g., an antibody or IgGsolution).

In a second aspect, a method of separating or purifying a negativelycharged material is described. The method includes forming a macroporousanion exchange resin, contacting the macroporous anion exchange resinwith negatively charged material, and adsorbing at least a portion ofthe negatively charged material on the macroporous anion exchange resin.The macroporous anion exchange resin is formed by preparing an aqueousphase composition that contains (a) a monomer mixture; (b) awater-soluble alkylene oxide or polyalkylene oxide porogen of Formula I;and (c) an aqueous phase solvent mixture that includes water and amono-alcohol having 1 to 4 carbon atoms. The mono-alcohol is present inan amount of at least 20 volume percent based on the volume of theaqueous phase solvent mixture. The monomer mixture includes (1) acrosslinking monomer containing N,N′-alkylenebis(meth)acrylamide,N,N′-heteroalkylenebis(meth)acrylamide or a combination thereof; and (2)a positively charged ionic monomer. Substantially all of the monomers inthe monomer mixture have a lipophilicity index less than or equal to 20.The formation of the macroporous anion exchange resin further includessuspending the aqueous phase composition in a non-polar organic solvent,polymerizing the monomer mixture to form particles of polymericmaterial, and removing the porogen from the particles.

A sample containing negatively charged materials can be contacted withan anion exchange resin at a pH where the anion exchange resin haspositively charged groups (e.g., at a pH of 1 to 10). In general, inorder to get effective adsorption of the negatively charged material tothe anion exchange resin, a pH of at least about 1 to 2 pH units abovethe pK of the material (or pI for a protein) can be used. To release theadsorbed material from the anion exchange resin, if desired, the pH canbe lowered at least 1 to 2 pH units, or more. Alternatively, when thecharged material is a biomolecule, the sample can be contacted with theanion exchange resin in a low ionic strength buffer (e.g., a 5 to 20millimolar buffer salt) at an appropriate pH (e.g., at a pH of about 6-8for BSA). To release the adsorbed biomolecule, a high ionic strengthbuffer is contacted with the anion exchange resin. In some embodiments,the high ionic strength buffer includes that same buffer compositionused to adsorb the material plus 1 molar sodium chloride. The adsorptionand release processes are typically performed at temperatures near roomtemperature.

Buffer salts useful for controlling pH include, but are not limited to,sodium phosphate, sodium carbonate, sodium bicarbonate, sodium borate,sodium acetate, and TRIS (tris(hydroxymethyl)aminomethane). Othersuitable buffers include “Good's” buffers such as MOPS(3-morpholinopropanesulfonic acid), EPPS(4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid), MES(2-morpholinoethanesulfonic acid), and others.

Some samples include a biological molecule that can be separated fromthe other sample constituents or that can be purified. Suitablebiological molecules include, for example, proteins, enzymes, vaccines,DNA, and RNA. Adjusting the pH of the sample can alter the charge ofsome biological molecules.

In a third aspect, a method of preparing a chromatographic column isdescribed. The method includes forming a macroporous anion exchangeresin and placing the macroporous anion exchange resin in a column. Themacroporous anion exchange resin is formed by preparing an aqueous phasecomposition that contains (a) a monomer mixture; (b) a water-solublealkylene oxide or polyalkylene oxide porogen of Formula I; and (c) anaqueous phase solvent mixture that includes water and a mono-alcoholhaving 1 to 4 carbon atoms. The mono-alcohol is present in an amount ofat least 20 volume percent based on the volume of the aqueous phasesolvent mixture. The monomer mixture includes (1) a crosslinking monomercontaining N,N′-alkylenebis(meth)acrylamide,N,N′-heteroalkylenebis(meth)acrylamide or a combination thereof; and (2)a positively charged ionic monomer. Substantially all of the monomers inthe monomer mixture have a lipophilicity index less than or equal to 20.The formation of the macroporous anion exchange resin further includessuspending the aqueous phase composition in a non-polar organic solvent,polymerizing the monomer mixture to form particles of polymericmaterial, and removing the porogen from the particles.

The anion exchange resin particles prepared by the method describedherein tend to be fairly rigid and have the mechanical strength neededfor use in chromatographic columns of any suitable dimension and underany suitable flow rates and pressure conditions. The particles can beused, for example, in a chromatographic column with high flow rates. Theanion exchange resins are suitable for use under the differentialpressure conditions that are commonly encountered in chromatographiccolumns. As used herein, the term “differential pressure” refers to thepressure drop across a chromatographic column. For example,chromatographic columns used for the downstream purification orseparation of therapeutic proteins can be used with superficialvelocities (e.g., flow rates) such as at least 150 cm/hr, at least 250cm/hr, at least 500 cm/hr, or at least 700 cm/hr to increaseproductivity. Faster flow rates typically lead to higher productivity.

In small chromatographic columns (e.g., columns with a diameter lessthan about 5 cm), the packed bed of anion exchange resin is wellsupported by the column wall. In such columns, anion exchange resinshaving a relatively wide range of rigidity can withstand differentialpressures in excess of 200 psi (1,380 kPa). However, in largechromatographic columns (e.g., columns with a diameter greater thanabout 5 cm), the packed bed of anion exchange resin has less supportfrom the column wall (e.g., a smaller fraction of the resin is incontact with the wall surfaces of the column). In such columns, anionexchange resins with higher rigidity tend to be able to withstanddifferential pressures of at least 25 psi (173 kPa). Some anion exchangeresins can withstand a differential pressure of 50 psi (345 kPa) to 200psi (1,380 kPa).

Suitable columns are known in the art and can be constructed of suchmaterials as glass, polymeric material, stainless steel, titanium andalloys thereof, or nickel and alloys thereof. Methods of filling thecolumn to effectively pack the anion exchange resin in the column areknown in the art.

The chromatographic columns can be part of an analytical instrument suchas a liquid chromatograph. When packed with the anion exchange resin,the chromatographic column can be used to separate an ionic materialfrom non-ionic materials or to separate one ionic material from anotherionic material with a different charge density. The amount of the ionicmaterial in the sample can be determined.

The chromatographic columns can be part of a preparative liquidchromatographic system to separate or purify an ionic material. Thepreparative liquid chromatographic system can be a laboratory scalesystem, a pilot plant scale system, or an industrial scale system.

In a fourth aspect, a method of preparing a filtration element isdescribed. The method includes forming a macroporous anion exchangeresin and disposing the macroporous anion exchange resin on a surface ofa filtration medium. The macroporous anion exchange resin is formed bypreparing an aqueous phase composition that contains (a) a monomermixture; (b) a water-soluble alkylene oxide or polyalkylene oxideporogen of Formula I; and (c) an aqueous phase solvent mixture thatincludes water and a mono-alcohol having 1 to 4 carbon atoms. Themono-alcohol is present in an amount of at least 20 volume percent basedon the volume of the aqueous phase solvent mixture. The monomer mixtureincludes (1) a crosslinking monomer containingN,N′-alkylenebis(meth)acrylamide, N,N′-heteroalkylenebis(meth)acrylamideor a combination thereof; and (2) a positively charged ionic monomer.Substantially all of the monomers in the monomer mixture have alipophilicity index less than or equal to 20. The formation of themacroporous anion exchange resin further includes suspending the aqueousphase composition in a non-polar organic solvent, polymerizing themonomer mixture to form particles of polymeric material, and removingthe porogen from the particles.

The filter element can be positioned within a housing to provide afilter cartridge. Suitable filtration medium and systems that include afilter cartridge are further described, for example, in U.S. Pat. No.5,468,847 (Heilmann et al.), incorporated herein by reference. Such afilter cartridge can be used to purify or separate biomolecules. In thisaspect, less rigid particles or smaller macroporous particles can beutilized than in the chromatographic column format due to the lowerpressure drops inherent in the filter cartridge system.

The filtration medium can have a single filtration layer or multiplefiltration layers and can be prepared from glass or polymeric fibers(e.g., polyolefin fibers such as polypropylene fibers). In someembodiments, the filtration medium includes a coarse pre-filtrationlayer and one or more finer filtration layers. For example, thefiltration medium can include a coarse pre-filtration layer and then aseries of additional filtration layers with progressively smalleraverage pore sizes. The anion exchange resin can be positioned on thelayer of the filtration medium having the smallest average pore size.

Selection of the pore size of the filtration medium depends on the sizeof the anion exchange resin. Typically the pore size of the filtrationmedium is selected to be smaller than the average diameter of the anionexchange resin. However, a portion of the anion exchange resin canpenetrate into the filtration medium.

The filtration medium can be in the form of vertical pleated filterssuch as those described in U.S. Pat. No. 3,058,594 (Hultgren). In otherembodiments, the filtration medium is in the form of horizontal,compound radially pleated filters such as those described in U.S. Pat.No. 4,842,739 (Tang et al.), incorporated herein by reference. Ahorizontal arrangement of the pleats can be desirable in applicationswhere a filter cartridge containing the filtration medium is used in thevertical direction. Such an arrangement can reduce the loss of the anionexchange resin from the filter element during use and storage.

In a fifth aspect, a method of making a porous composite material isdescribed. The method includes forming a macroporous anion exchangeresin and incorporating the macroporous anion exchange resin in acontinuous, porous matrix. The macroporous anion exchange resin isformed by preparing an aqueous phase composition that contains (a) amonomer mixture; (b) a water-soluble alkylene oxide or polyalkyleneoxide porogen of Formula I; and (c) an aqueous phase solvent mixturethat includes water and a mono-alcohol having 1 to 4 carbon atoms. Themono-alcohol is present in an amount of at least 20 volume percent basedon the volume of the aqueous phase solvent mixture. The monomer mixtureincludes (1) a crosslinking monomer containingN′N′-alkylenebis(meth)acrylamide, N,N′-heteroalkylenebis(meth)acrylamideor a combination thereof; and (2) a positively charged ionic monomer.Substantially all of the monomers in the monomer mixture have alipophilicity index less than or equal to 20. The formation of themacroporous anion exchange resin further includes suspending the aqueousphase composition in a non-polar organic solvent, polymerizing themonomer mixture to form particles of polymeric material, and removingthe porogen from the particles.

The continuous, porous matrix is typically a woven or non-woven fibrousweb, porous fiber, porous membrane, porous film, hollow fiber, or tube.Suitable continuous, porous matrixes are further described in U.S. Pat.No. 5,993,935 (Rasmussen et al.), incorporated herein by reference.

A continuous, porous matrix that is a fibrous web can provide suchadvantages as, for example, large surface area, ease of manufacture, lowmaterial cost, and a variety of fiber textures and densities. Although awide range of fiber diameters is suitable, the fibers often have anaverage diameter of 0.05 micrometers to 50 micrometers. The webthickness can be varied to fit the end use application (e.g., about 0.2micrometers to about 100 cm).

The composite material can be prepared, for example, using melt-blowingmethods. For example, a molten polymeric material can be extruded toproduce a stream of melt blown fibers. The anion exchange resin can beintroduced into the stream of fibers and intermixed with the fibers. Themixture of fibers and anion exchange resin can be collected on a screensuch that a web is formed. The anion exchange resin can be dispersedwithin the fibrous web. In some embodiments, the anion exchange resincan be dispersed uniformly throughout the fibrous web.

The composite material can also be prepared with a fibrillated polymermatrix such as fibrillated polytetrafluoroethylene (PTFE). Suitablemethods are more fully described in U.S. Pat. Nos. 4,153,661 (Ree etal.); 4,565,663 (Errede et al.); 4,810,381 (Hagen et al.); and 4,971,736(Hagen et al.), all of which are incorporated herein by reference. Ingeneral, these methods involve blending the anion exchange resin with apolytetrafluoroethylene dispersion to obtain a putty-like mass,subjecting the putty-like mass to intensive mixing at a temperature of5° C. to 100° C. to cause fibrillation of the PTFE, biaxiallycalendaring the putty-like mass, and drying the resultant sheet.

In another method of preparing the composite material, the anionexchange resin can be dispersed in a liquid and then blended with athermoplastic polymer at a temperature sufficient to form a homogenousmixture. The homogeneous mixture can be placed in a mold having adesired shape. Upon cooling of the mixture, the liquid can be phaseseparated leaving a thermoplastic polymeric matrix that containsdispersed anion exchange resin particles. This method is furtherdescribed in U.S. Pat. No. 4,957,943 (McAllister et al.), incorporatedherein by reference.

The amount of anion exchange resin incorporated into the continuous,porous matrix is at least 1 volume percent, at least 5 volume percent,at least 10 volume percent, at least 20 volume percent, at least 30volume percent, at least 40 volume percent, or at least 50 volumepercent based on the volume of the resulting composite. The amount ofanion exchange resin incorporated into the continuous, porous matrix cancontain up to 99 volume percent, up to 95 volume percent, up to 90volume percent, up to 85 volume percent, or up to 80 volume percentbased on the volume of the resulting composite. Composites having alarger amount of anion exchange resin tend to have a larger anionexchange capacity.

The foregoing describes the invention in terms of embodiments foreseenby the inventor for which an enabling description was available,notwithstanding that insubstantial modifications of the invention, notpresently foreseen, may nonetheless represent equivalents thereto.

EXAMPLES

These examples are merely for illustrative purposes only and are notmeant to be limiting on the scope of the appended claims. All parts,percentages, ratios, and the like in the examples are by weight, unlessnoted otherwise. Solvents and other reagents used were obtained fromSigma-Aldrich Chemical Company (Milwaukee, Wis.) unless otherwise noted.

Test Methods Anion Exchange Capacity

A 50 volume percent slurry of anion exchange beads in DI (deionized)water was prepared by mixing the beads with water, centrifuging themixture at 3,000 relative centrifugal force (rcf) for 20 minutes, andthen adjusting the amount of water so that the total volume was twicethat of the packed bead bed. The slurry was mixed well to suspend thebeads, then a 400 microliter sample of the slurry was pipetted into a 5milliliter cellulose acetate CENTREX MF centrifugal micro-filter havinga pore size of 0.45 micrometer. The micro-filter was obtained fromSchleicher & Schuell through VWR, Eagan, Minn. The water was removedfrom the slurry on the micro-filter by centrifugation at 3,000 rcf for 5minutes. A solution of 4 mL of 10 mM MOPS at pH 7.5 was mixed with thebeads on the micro-filter. The liquid was removed by centrifugationagain at 3,000 rcf for 10 minutes. The filtrates were discarded. A 4.5mL sample of BSA (about 9 mg/mL) (commercially available fromSigma-Aldrich, St. Louis, Mo.) in the same MOPS buffer was added to themicro-filter containing the beads. The mixture was tumbled overnight andthen the supernatant was removed from the beads by centrifugation at3,000 rcf for 15 minutes.

The filtrate was analyzed by UV spectroscopy, comparing the absorbanceat 280 nm to that of the starting BSA solution; the difference was usedto calculate the BSA anion exchange capacity of the beads. Assays wererun in triplicate and averaged.

Surface Area and Porosity Measurements

Approximately 0.1-1.0 g of each sample was transferred to a 1.3centimeter (0.5 inch) diameter sample tube available from Micromeritics,Inc. of Norcross, Ga. and degassed using a system commercially availablefrom Micromeritics under the trade designation VACPREP 061 for 24 hoursat 100° C. under vacuum (below 10 mTorr or 0.015 mbar). After degassing,the samples were allowed to cool for 10 minutes under vacuum at ambienttemperature (i.e., 20° C. to 25° C.), and then loaded onto a surfacearea and porosity analyzer commercially available from Micromeriticsunder the trade designation TRISTAR 3000.

A 45 point adsorption/40 point desorption isotherm was set up withrelative pressures (P/P_(o)) starting at about 0.0 up to about 1.0 witha tighter distribution of points between 0.95 and 1.0 (See Table forTarget Pressures and Points). No first “pressure fixed dose” was set.The maximum volume increment was set at 10.00 cc/g at STP, the “absolutepressure tolerance” was set at 5 mm Hg, and the “relative pressuretolerance” was set at 2.0%. “Fast evacuation” and “leak test” optionswere not used. With the dewar of liquid nitrogen lowered (i.e., thesample was not in the liquid nitrogen), an evacuation time of 0.5 hourswas implemented during the free space measurement. The dewar was raisedfor analysis (i.e., the tube containing the sample was placed in liquidnitrogen). At 77.350 K (the temperature of liquid nitrogen), P_(o) wasmeasured at 120 min intervals during the analysis. The gas adsorptiveproperties using a standard Pstat versus temperature table for nitrogengas were set at the following values: non-ideality factor, 0.0000620;density conversion factor, 0.0015468; molecular cross-sectional area,0.162 nm₂. BJH adsorption/desorption cumulative pore volumes andcumulative surface areas were calculated for pores between 17 Å-2,000 Ådiameter (corresponding to pores between 2 and 200 nanometers), andbased on quantity of N₂ adsorbed at each relative pressure during the 45adsorption points and 40 desorption points.

The following table indicates the adsorption and desorption points usedfor the analysis. The cumulative surface area and cumulative pore volumeduring adsorption are reported below for various examples.

Table of Target Pressures and Points Relative Pressure BJH BJH Point(P/Po) Adsorption Desorption 1 0.060 X 2 0.080 X 3 0.120 X 4 0.140 X 50.160 X 6 0.200 X 7 0.250 X 8 0.300 X 9 0.350 X 10 0.400 X 11 0.450 X 120.500 X 13 0.550 X 14 0.600 X 15 0.650 X 16 0.700 X 17 0.740 X 18 0.770X 19 0.800 X 20 0.820 X 21 0.840 X 22 0.860 X 23 0.875 X 24 0.890 X 250.905 X 26 0.915 X 27 0.925 X 28 0.933 X 29 0.940 X 30 0.947 X 31 0.953X 32 0.959 X 33 0.964 X 34 0.968 X 35 0.971 X 36 0.974 X 37 0.977 X 380.980 X 39 0.982 X 40 0.984 X 41 0.986 X 42 0.988 X 43 0.989 X 44 0.990X 45 1.000 46 0.990 X 47 0.989 X 48 0.988 X 49 0.986 X 50 0.984 X 510.982 X 52 0.980 X 53 0.977 X 54 0.974 X 55 0.971 X 56 0.968 X 57 0.964X 58 0.959 X 59 0.953 X 60 0.947 X 61 0.940 X 62 0.933 X 63 0.925 X 640.915 X 65 0.905 X 66 0.890 X 67 0.875 X 68 0.860 X 69 0.840 X 70 0.820X 71 0.800 X 72 0.770 X 73 0.740 X 74 0.700 X 75 0.650 X 76 0.600 X 770.550 X 78 0.500 X 79 0.450 X 80 0.400 X 81 0.350 X 82 0.300 X 83 0.250X 84 0.200 X 85 0.140 X

Table of Abbreviations Abbreviation or Trade Designation Description MBAN,N′-methylenebisacrylamide MAPTAC[3-(methacryloylamino)propyl]trimethylammonium chloride used as a 50percent by weight solution in water. APTAC(3-acrylamidopropyl)trimethylammonium chloride used as a 75 percent byweight solution in water DMAPMAN-[3-(dimethylamino)propyl]methacrylamide DMA N,N-dimethylacrylamideTMEDA N,N,N′,N′-tetramethylethylenediamine. BSA Bovine serum albumin.PEG 400 Polyethyleneglycol with hydroxy end groups having an averagemolecular weight 400 g/mole PEG 600 Polyethylene glycol with hydroxy endgroups, average molecular weight 600 g/mole PEG 1000 Polyethylene glycolwith hydroxy end groups, average molecular weight 1,000 g/mole PEG 2000Polyethylene glycol with hydroxy end groups, average molecular weight2,000 g/mole PEG 3400 Polyethylene glycol with hydroxy end groups,average molecular weight 3,400 g/mole IPA Isopropanol

Example 1

A 50:50 by weight MAPTAC/MBA copolymer was prepared by reverse-phasesuspension polymerization using a polyethylene glycol porogen (PEG 400)in the aqueous phase.

A polymeric stabilizer (0.28 grams), toluene (132 mL), and heptane (243mL) were added to a flask equipped with a mechanical stirrer (stirringrate 450 rpm), nitrogen inlet, thermometer, heating mantel withtemperature controller, and condenser. The polymeric stabilizer was a91.8:8.2 by weight copolymer of isooctylacrylate and2-acrylamidoisobutyramide (prepared as described in Rasmussen, et al.,Makromol. Chem., Macromol. Symp., 54/55, 535-550 (1992)). Thenon-aqueous solution in the flask was heated to 35° C. with stirring,and sparged with nitrogen for 15 minutes.

An aqueous solution was prepared that contained MBA (7.0 grams), MAPTAC(14.0 grams of a 50 percent by weight aqueous solution), methanol (35mL), PEG 400 (15 mL), and deionized water (38 mL). This second solutionwas stirred and heated at 30° C. to 35° C. to dissolve the MBA. Sodiumpersulfate (1.12 grams dissolved in 5 mL deionized water) was added tothe second solution with additional stirring. The aqueous solution wasadded to the reaction flask containing the non-aqueous solution. Theresulting mixture was stirred and nitrogen sparged for 5 minutes.

TMEDA (1.12 mL) was added to initiate the polymerization. The reactiontemperature quickly rose to 42.6° C., then slowly subsided. The reactionmixture was stirred for a total of 2.3 hours from the time of TMEDAaddition, filtered using a sintered glass funnel, washed with acetone(2×250 mL), methanol (2×250 mL), and acetone (1×250 mL) and dried atroom temperature under vacuum to yield 15.8 grams of colorlessparticles.

Based on scanning electron micrographs, the resin was in the form ofspherical particles ranging from about 20 to 200 micrometers indiameter. The protein anion exchange capacity was measured according tothe procedure described in the Test Methods using BSA as the protein.The data is shown in Table 1.

Prior to porosity measurements, the resin was subjected to additionalwashing to remove any remaining porogen. Approximately 5 mL of the resinparticles were mixed with 40 mL of 1:1 solution of acetonitrile andwater based on volume. The mixture was tumbled for 3 days. The samplewas filtered, washed with acetone (3×100 mL), and then dried undervacuum. Surface area and porosity were measured as described in the TestMethods. The data is shown in Table 1.

Comparative Example Cl

Anion exchange beads were prepared as described in Example 1, exceptthat the PEG 400 (porogen) was replaced by methanol in the aqueoussolution. Anion exchange capacity for the protein BSA is shown in Table1.

Example 2

For Example 2, the same procedure used for Example 1 was followed exceptthat the amounts of each monomer (MBA and MAPTAC) were doubled. Anionexchange capacity for the protein BSA and porosity measurements areshown in Table 1.

Example 3

For Example 3, the same procedure used for Example 1 was followed exceptthat the amounts of each monomer (MBA and MAPTAC) were doubled, the onlysolvent in the non-aqueous phase was heptane (536 mL), IPA (80 mL) wasused instead of methanol, and the total aqueous phase was 150 mL. Anionexchange capacity for the protein BSA and porosity measurements areshown in Table 1.

Example 4

For Example 4, the same procedure used for Example 1 was followed exceptthat the total amount of monomers was doubled, and the monomers wereMBA, MAPTAC, and DMA. The amount of each monomer in the monomer mixturewas 35 weight percent MBA, 30 weight percent MAPTAC, and 35 weightpercent DMA. Anion exchange capacity for the protein BSA and porositymeasurements are shown in Table 1.

Example 5

For Example 5, the same procedure used for Example 4 was followed exceptthat the only solvent in the non-aqueous phase was heptane (536 mL) andIPA was used in place of methanol. Anion exchange capacity for theprotein BSA and porosity measurements are shown in Table 1.

Example 6

For Example 6, the same procedure used for Example 1 was followed exceptthat the total amount of monomers was doubled. The amount of eachmonomer in the monomer mixture was 70 weight percent MBA and 30 weightpercent MAPTAC. Anion exchange capacity for the protein BSA and porositymeasurements are shown in Table 1.

Example 7

For Example 7, the same procedure used for Example 6 was followed exceptthat the only solvent in the non-aqueous phase was heptane (536 mL), IPA(40 mL) was used in place of methanol, and the total aqueous phase was110 mL. Anion exchange capacity for the protein BSA and porositymeasurements are shown in Table 1.

TABLE 1 Anion BJH Exchange Cumulative BJH Capacity Pore Cumulative forBSA Volume Surface Area Example (mg/mL) (cc/g) (m²/g) C1 10 NM NM 1 720.68 174 2 79 0.71 192 3 30 0.17  42 4 97 0.51 137 5 86 0.65 152 6 860.69 221 7 46 0.59 176 NM = not measured

Examples 8-12

For Examples 8-12, the same procedure used for Example 2 was followedexcept that the molecular weight of the PEG was varied as indicated inTable 2 and the total polymerization time was reduced to 1 hour afteraddition of the TMEDA. Anion exchange capacity for the protein BSA andporosity measurements are listed in Table 2.

The scanning electron micrograph of the surface of Example 9 is shown inFIG. 1 at a magnification of 15,000×. FIG. 2 shows a scanning electronmicrograph of the surface of Example 11 at a magnification of 15,000×.

TABLE 2 Anion Exchange BJH BJH Capacity Cumulative Cumulative for BSASurface Area Pore Volume Example PEG (mg/mL) (m²/g) (cc/g) 8 400 68 1240.49 9 600 95 93 0.46 10 1000 66 87 0.36 11 2000 8 2 0.01 12 3400 12 40.02

Example 13

For Example 13, the same procedure as used in Example 6 was used, exceptthat APTAC was used in place of MAPTAC, the only solvent in thenon-aqueous phase was heptane (536 mL), IPA (95 mL) was used in place ofmethanol and the total aqueous phase was 160 mL. Anion exchange capacityfor BSA was 46 mg/mL.

Example 14

A 65:35 by weight DMAPMA/MBA copolymer was prepared by the generalprocedure of Example 1 using PEG 400 as porogen in the aqueous phase.The only solvent in the non-aqueous phase was heptane (536 mL). Theaqueous phase consisted of MBA (9.8 g), DMAPMA (18.2 g), glacial aceticacid (6.15 ml), PEG 400 (15 ml), IPA (95 ml), DI water (43.85 ml), andsodium persulfate (0.55 g). Anion exchange capacity for BSA was 40mg/mL.

Comparative Example C2

For Comparative Example C2, the procedure of Example 16 was repeatedexcept that the PEG 400 was replaced with IPA (15 ml). Anion exchangecapacity for BSA was 3 mg/mL.

1. A method of separating or purifying a negatively charged material,the method comprising: preparing a macroporous anion exchange resin, thepreparing comprising forming an aqueous phase composition comprising a)a monomer mixture comprising i) a crosslinking monomer comprisingN,N′-alkylenebis(meth)acrylamide,N,N′-heteroalkylenebis(meth)acrylamide, or a combination thereof in anamount in a range of 15 to 90 weight percent based on a weight ofmonomers in the monomer mixture; and ii) a positively charged ionicmonomer, wherein substantially all of the monomers in the monomermixture have a lipophilicity index less than or equal to 20; and b) awater-soluble porogen of Formula IR¹—(R²—O)_(n)—R³  (I) wherein R¹ is hydroxy, alkoxy, carboxy, acyloxy,or halo; each R² is independently an alkylene having 1 to 4 carbonatoms; R³ is hydrogen, alkyl, carboxyalkyl, acyl, or haloalkyl; n is aninteger of 1 to 1,000; and c) an aqueous phase solvent mixturecomprising water and a mono-alcohol having 1 to 4 carbon atoms, whereinthe mono-alcohol is present in an amount of at least 20 weight percentbased on a total weight of the aqueous phase solvent mixture; suspendingthe aqueous phase composition in a non-polar organic solvent;polymerizing the monomer mixture to form particles of polymericmaterial; and removing the porogen from the particles to form themacroporous anion exchange resin; contacting the macroporous anionexchange resin with negatively charged material; and adsorbing at leasta portion of the negatively charged material on the macroporous anionexchange resin.
 2. The method of claim 1, wherein the positively chargedionic monomer is of Formula II or a quaternary ammonium salt thereof

wherein R⁴ is hydrogen or methyl; L is oxy or —NH—; Y is an alkylene;and each R⁵ is independently hydrogen, alkyl, hydroxy alkyl, aminoalkyl, or the two R⁵ groups taken together with the nitrogen atom towhich they are attached form a heterocyclic group that is aromatic,partially unsaturated, or saturated, wherein the heterocyclic group canoptionally be fused to a second ring that is aromatic, partiallyunsaturated, or saturated.
 3. The method of claim 1, wherein thenegatively charged material is a biological molecule.
 4. The method ofclaim 3, wherein the biological molecule is a protein.
 5. The method ofclaim 1, further comprising placing the macroporous anion exchange resinin a column.
 6. The method of claim 1, further comprising disposing themacroporous anion exchange resin on a surface of a filtration medium. 7.The method of claim 1, further comprising incorporating the macroporousanion exchange resin in a continuous, porous matrix.
 8. The method ofclaim 1, wherein the macroporous anion exchange resin has at least somepores having a diameter less than or equal to 200 nanometers.
 9. Themethod of claim 8, wherein the macroporous anion exchange resin has apore volume of at least 0.10 cubic centimeters per gram and a surfacearea of at least 20 m²/g.
 10. The method of claim 1, wherein thecrosslinking monomer is present in an amount of 25 to 75 weight percentbased on a total weight of monomers in the monomer mixture and thepositively charged ionic monomer is present in an amount of 25 to 75weight percent based on the total weight of the monomers in the monomermixture.
 11. The method of claim 1 wherein R² is ethylene or propylene.12. The method of claim 1, wherein R¹ is hydroxy and R³ is hydrogen orR¹ is hydroxy and R³ is alkyl.
 13. The method of claim 1, wherein theporogen comprises a polyethylene oxide with a molecular weight nogreater than 1,000 g/mole.
 14. The method of claim 13, wherein thepolyethylene oxide has hydroxy end groups.
 15. The method of claim 1,wherein the porogen comprises a mixture of a first porogen comprising analkylene glycol and a second porogen comprising a polyalkylene oxide.16. The method of claim 15, wherein the first porogen comprises ethyleneglycol and the second porogen comprises polyethylene oxide with hydroxyend groups.
 17. The method of claim 1, wherein the mono-alcohol ispresent in an amount of at least 50 percent based on the total weight ofthe aqueous phase solvent mixture.